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What happens when several scientific disciplines join forces to tackle one of the biggest challenges in drug discovery and development? You get “a fast-moving field with a lot of innovation and a lot of exciting research,” says Richard Eglen, Ph.D., of Corning Life Sciences, former SLAS president and a guest editor of the June 2017 SLAS DiscoverySpecial Issue on3D Cell Culture, Drug Screening and Optimization.

Eglen believes SLAS and the SLAS journals “are perfectly positioned to be in this wave of exciting science. They have always been a forum for multi-disciplinary areas like this.” He and co-guest editor Jean-Louis Klein, Ph.D., of GlaxoSmithKline have collected 17 outstanding peer-reviewed papers that highlight important new advances in 3D culture technology and its use in drug development. Thanks to the generosity of Corning Life Sciences, free online access to select reports is available.

The emerging interest in 3D cell culture was evident at SLAS2017 in Washington, DC. According to Eglen, “The buzz around 3D culture was noticeable. There were several presentations and posters around 3D cell culture from different companies and different disciplines. We’re now seeing a convergence of an interest in phenotypic screening with an interest in having cells in a condition that closely mimics how they grow naturally.”

This is due in part to a renewed interest in phenotypic drug screening. As Klein explains, “Until recently researchers would start with a screen and adapt the assay to the screen to make sure it was a robust assay.” But this approach contributed to the high attrition rate of compounds in clinic. “The models didn’t accurately predict what drugs do in humans. If we want to improve the success rate in clinic, we must have models that are relevant in human disease.”

Ideally, these models will not only mimic human biology but also will be reproducible and adaptable to high-throughput screening (HTS) and automation. While the use of cellular assays in drug discovery isn’t new, as the editors point out, it has historically been limited to cells grown on two-dimensional flat, glass or plastic planar surfaces. “This works well with automation and instrument detection systems, but unfortunately, cells don’t only grow in two dimensions,” Eglen says.

Early attempts at 3D cultures were labor-intensive. Eglen says one of the first techniques was the hanging drop method, where cells come together within a drop of fluid hanging from a cover to form a ball of cells called a spheroid. “Those are hard to do on a reproducible basis, and very difficult to take into an automation system. They're not robotic-friendly.”

Evolving Technology

As several papers in the SLAS Discovery special issue illustrate, the technology is evolving to overcome some of these limitations. For example, Madoux and colleagues describe using customized 1536-well plates with an ultra-low-attachment surface and round-bottom wells to allow the consistent formation, size monitoring and viability assessment of 3D spheroids that can then be used to screen a large-compound library for cytotoxic effects. Free online access to this paper is sponsored by Corning Life Sciences.

Meanwhile, Thakuri and Tavana at the University of Akron, OH, are taking a different approach. Their paper describes generating a spheroid within an aqueous drop immersed in a second, immiscible aqueous phase. Using robotics to facilitate the formation, maintenance and drug treatment of colon cancer spheroids, they demonstrate the feasibility of HTS with this method using 25 anticancer compounds.

In the paper entitled “RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions,” Fu and colleagues at the National Institutes of Health (NIH), Rockville, MD, examine the differences in tumor growth of breast cancer and colon cancer cells grown in a 3D culture compared to 2D cultures. They develop a HTS assay to generate 3D spheroids in ultra-low-attachment 384-well plates and a corresponding 2D monolayer culture assay in 384-well flat-bottom plates. Using these models, they are able to detect subtle differences in 2D versus 3D tumor growth driven by siRNA-mediated gene silencing. Free online access to this paper is available due to the Corning Life Sciences sponsorship of the special issue.

Is Three Greater than Two?

Designing automated HTS 3D assays is just the first step. As Eglen points out, it raises the question “If you get different results when you look at the cells in three dimensions versus two dimensions, is one better at mimicking the situation in the patient than the other, or are they just different?” Klein agrees: “We can’t assume that if we use a spheroid or an extracellular matrix gel, it is actually better than a cell line grown in 2D. The next step is to show it is a better model than 2D.”

In their paper “An Automated Multiplexed Hepatotoxicity and CYP Induction Assay Using HepaRG Cells in 2D and 3D,” Ott and Ramachandran at the University of Kansas Medical Center in Kansas City, KS, present a clear advantage of 3D assays over 2D assays in detecting liver-toxic drugs. They compare the ability of a 3D cell culture assay and a 2D cell culture assay to detect known drug-induced liver injury positive and negative compounds. The 3D HepaRG model had a 60-70 percent sensitivity at 24 hours and 7 days, while the 2D model had a 50-60 percent sensitivity. Moreover, the 3D model correctly identified 86 percent of the most toxic compounds, while the 2D model only identified 57 percent.

Selby and colleagues from the Frederick National Lab for Cancer Research in Frederick, MD, compare a 3D assay to a 2D assay to illustrate that the tumor microenvironment also plays a key role in response to therapy. They describe differences observed in cell viability and sensitivity to certain oncology compounds when cells are grown as spheroids versus conventional monolayer 2D cultures. After optimizing the NCI60 cell line panel for generating 3D spheroids of a prespecified diameter (300–500 μm) in ultra-low attachment (ULA) plates, the authors use the data to compare predictive power of the 3D models versus 2D monolayer cultures. They conclude that predictive assays may require co-cultures with multiple cell types. Free online access to this paper is sponsored by Corning Life Sciences.

New Materials: Hydrogels and Microfluidics

Advances in the materials used for designing 3D models have allowed for this type of exploration. For instance, Lal-Nag and colleagues at NIH and the University of Chicago examine the effects of different drugs on cells grown in a 3D microenvironment culture model of the omentum compared to cells grown on plastic, in monolayers and in spheroids. Not surprisingly, the efficacy of the drugs was different in the different models, underlining the importance of screening drugs in model systems that mimic both the tumor microenvironment and the cell adhesion mode of cancer cells. Free online access to this paper also is available through Corning Life Sciences.

Two other papers in the issue also explore the usefulness of hydrogels in 3D models. Zhu and Ding examine the effect of viral infection and corresponding treatment on the pattern formation of cells in a 3D hydrogel model. Separately, Zhang and colleagues describe how a pre-cast synthetic hydrogel can be adapted and simplified for use in a variety of 3D cell cultures, increasing its versatility.

Eglen sees the use of microfluidics and hydrogels coming to the fore as the areas of organoids and organ-on-chip start to develop. “With organ-on-a-chip technology you get the dynamic of flow, that is fluid moving through the chip. With cells grown in channels through which the fluid moves, you can start to mimic tissues perfused by blood.”

Advances in Imaging Technology

Another area of advancing technology is cell imaging. At Nexcelom Bioscience LLC, Lawrence, MA, Cribbes and colleagues describe the development of a novel multiparametric drug-scoring system for 3D multicellular tumor spheroids using fluorescent staining and the Celigo Image Cytometer. They show how the data gathered with this technique can be used to delineate differences between drugs that induce cytotoxic and cytostatic effects and therefore identify and classify potential drug candidates earlier in the drug discovery process.

Meanwhile, Lal-Nag and colleagues are using advanced imaging techniques to measure spheroid growth, cell viability and cell death in real time. Using the data from these assays, they are able to monitor tumor formation and long-duration drug dosing that mimic such dosing regimens in vivo. Free online access to their paper is available through the support of Corning Life Sciences.

A Role for Everyone

Klein says he is very excited right now about cell biology in general. “It’s in a new phase because of all these new techniques. The next five years will be fantastic for this field, no doubt about that.”

What’s the next step in moving the science forward? Both editors feel collaboration is key. “And not just between academia and biotech or pharma,” says Eglen, “but other disciplines as well. We’ve got cell biologists moving into this field who are looking for new ways to grow cells. We’ve got bioengineers designing different surfaces for the cells to grow on. We’ve got plastic engineers who'll design microfluidic chips or different vessels to growing the cells on. We’ve got physical technologists moving into this field with innovative ways to measure it.” Fang and Eglen’s reviewincluded in the special issue provides further detail and is available to all due to Corning’s sponsorship of the issue.

Klein agrees, “We need to bring the right people together, to talk together and work together.” He hopes that this special issue helps convey that message.

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